Assay for prions

ABSTRACT

The invention relates to a method for detection of abnormal PrP in a sample of blood or urine, said method comprising:
         (a) diluting the sample with buffer to comprise final concentrations of
           (i) 10 mM to 500 mM buffer agent;   (ii) 1% to 10% w/v bovine serum albumin; and   (iii) 1% to 8% w/v CHAPS;   
           (b) adding steel particles and incubating to allow PrP binding;   (c) washing the steel particles to remove diluted sample; and   (d) detecting abnormal PrP captured on the steel particles using antibody capable of binding said abnormal PrP.       

     The invention also provides compositions and kits.

RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 13/824,307, filed Mar. 15, 2013, which is a 371 of InternationalApplication No. PCT/GB2011/001341, filed Sep. 13, 2011, which claims thebenefit of Great Britain Application No. 1015569.5, filed Sep. 16, 2010,the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to an assay for detecting prions. In particularthe invention relates to an assay for detecting prion infection in bloodor urine, most suitably blood.

BACKGROUND TO THE INVENTION

Prion diseases are a group of fatal neurodegenerative disordersincluding variant Creutzfeldt-Jakob disease (vCJD) which originates fromexposure to bovine spongiform encephalopathy (BSE). Iatrogenictransmission from asymptomatic patients with vCJD prion infection viablood transfusion has had a major impact on public health policy.

Prion Disease encompasses a range of closely related and uniformly fatalneurodegenerative disorders affecting the central nervous system ofhumans and animals. They include Creutzfeldt-Jakob disease (CJD),Gerstmann-Striiussler-Scheinker disease (GSS), fatal familial insomnia(FFI) and kuru in humans, bovine spongiform encephalopathy (BSE) incattle, chronic wasting disease (CWD) of deer and Elk and scrapie insheep (1;2). The emergence of variant CJD (vCJD), and the confirmationthat it originates from exposure to bovine spongiform encephalopathy(BSE) (3;4) has raised a plethora of public health concerns adverselyaffecting surgery, dentistry, organ transplantation and bloodtransfusion.

Exposure of the UK population to BSE has been widespread with over181,000 cases confirmed to date in cattle (5) and yet the prevalence ofhuman infection in the UK has yet to be determined with accuracy. Aretrospective study of archived surgical lymphoreticular specimensestimated a prevalence of infection in the UK of 237 per million (95%confidence interval 49-692 per million)(6), far higher than the numberof vCJD clinical cases thus far with further studies failing to refinethese estimates (7). The demonstration of sub-clinical carrier states ofprion infection in animal models (8-10) offers a potential explanationfor the discrepancy between clinical cases and supports a prevalencewhich is likely to be in excess of 1 in 10,000 in the UK (11).

Concern regarding secondary transmission of vCJD has had extensiveeffects on public health policy in the UK and elsewhere. As theclinically silent incubation period of prion infection in humans can beprolonged, potentially exceeding 50 years (12), and the prevalence ispoorly defined, the extent of future transfusion-transmission of vCJDcannot be estimated or accurately risk-assessed. Secondary infectionfrom the clinically silent population has already been confirmed in fourrecipients of blood transfusion (13-15). Whilst the number oftransfusion recipients positively identified as having received vCJDcontaminated packed red cells is small, a much larger cohort of around7000 recipients of contaminated plasma products have already beenidentified and notified of their at-risk status (16). Concern for thiscohort has been heightened by post-mortem evidence for infection withvCJD prions in the spleen of a person with haemophilia (11).

The infectious agents, or prions, responsible for transmission ofdisease are composed principally if not entirely of a misfolded form ofthe host prion protein, PrP^(C). PrP^(C) is ubiquitous, althoughexpressed at highest levels in the central nervous system (CNS) andcells of the immune system. When recruited during prion propagationPrP^(C) is remodeled to an aggregated, detergent insoluble isoformdesignated PrP^(Sc) chemically identical but conformationally distinctfrom PrP^(C) (17). Detection of PrP^(Sc) in CNS and lymphoreticulartissues correlates widely with infection and the presence of prioninfectivity (18-20) and is accepted as 100% specific for prioninfection.

Although the quantities of PrP^(Sc) deposited in neural tissues aresufficient during the symptomatic phase of illness for detection byconventional immunoassays such as western blotting and ELISA, levels inperipheral tissues are significantly lower (21). Quantification ofinfectious titre using rodent models has indicated that the levels ofinfectivity, and by inference PrP^(Sc), in blood may be extremely low,with buffy coat fractions containing between 2-10 LD₅₀ Units ml⁻¹ duringthe asymptomatic phases of disease, rising to 100 LD₅₀ Units ml⁻¹ duringthe clinical stage (22;23).

In order to successfully identify infection in blood an assay must beable to detect PrP in a range which is several orders of magnitude belowthe sensitivity of conventionally employed immunoassays. Furthermore,the ratio of background PrP^(C), which is chemically identical toPrP^(Sc), is higher in blood than any other tissue and the high lipidand protein content of blood also contribute to non-specific backgroundsignals. Conventionally, immunoassays for PrP^(Sc) have been dependentupon protease pre-treatment of tissues to degrade PrP^(C) and otherproteins, thereby reducing cross-reactivity (24). It has now been shownin a number of different studies that the majority of disease associatedPrP may well be sensitive to proteolytic digestion with proteinase K(PK) (25-31).

The observation that prions can bind avidly to metal surfaces (32;33)has been used to develop quantitative assays for infectivity (34) thatapproach the high levels of sensitivity required to detect the lowlevels of prions and abnormal PrP associated with blood. However, priorart assays typically assess infectivity in an animal model or cellculture system which is labour intensive and time consuming as well asraising ethical issues about use of animals.

Current evidence clearly indicates there is risk of iatrogenic vCJD fromtransfusion of blood and purified blood components (13-15;36) and byinference from many forms of surgical and dental interventions. Takentogether with the knowledge that sub-clinical carrier states of priondisease can exist (8;9) alongside protracted incubation periods forclinical disease which may span several decades (12) the implicationsfor current and future public health are substantial. Current riskreduction strategies in the UK are extremely costly involvingleucodepletion of transfused packed red cells and the sourcing of plasmafrom the USA based on estimates of approximately 40% of infectivitybeing associated with leucocytes with the remainder in plasma (37).Studies modelling the use of leucocyte depletion have demonstratedreductions in blood infectivity of between 58% (37) and 72% (38). Theseand related issues also apply outside the UK, for example blood donationis not permitted in the USA if the prospective donor has spent acumulative period of 6 moths or more in Europe. This causes significantissues for US public and military blood transfusion services.

It remains a major problem that a non-invasive and specific test forprion infection is not available.

The invention seeks to overcome problem(s) associated with the priorart.

SUMMARY OF THE INVENTION

The invention provides a method for the capture and detection ofabnormal PrP from whole blood or urine using a solid-state matrix.However, rather than assay this material for infectivity by transfer tocell-culture or amplify the material for visualisation by westernblotting we have developed an assay for the direct immunodetection ofsurface-bound material, avoiding the use of any proteolytic processingto ensure the complete ensemble of abnormal PrP isoforms are availablefor detection.

Central to these methods are the provision of specific buffer systemsfor the dilution of whole blood or urine leading to the successfulcapture of abnormal PrP from the sample on substrate such as steelparticles.

Here we present the results of applying this assay to a blind panel of190 whole blood samples which contained 21 samples obtained from vCJDpatients, 100 normal control samples obtained from National BloodService and 69 neurological disease controls. We also present resultsfrom urine.

Provision of a blood or urine test which can identify asymptomaticpatients according to the present invention provides the ability tomanage the risk of transmission and allow early entry into therapeuticclinical trials.

Assay sensitivity was found to be several orders of magnitude higherthan any previously reported and was able to distinguish a 10⁻¹⁰dilution of exogenously spiked vCJD-infected brain from normal controlbrain with a significance of <0.0001. Analysis of the blind panel of 190samples indicated the assay has a sensitivity for the detection ofvCJD-infected patient blood of 71% and predicted overall specificity ofapproximately 99.97%.

Thus in one aspect the invention provides a method for detection ofabnormal PrP in a sample of blood or urine, said method comprising:

-   -   (a) diluting the sample with buffer to comprise final        concentrations of        -   (i) 10 mM to 500 mM buffer agent;        -   (ii) 1% to 10% w/v bovine serum albumin; and        -   (iii) 1% to 8% w/v CHAPS;    -   (b) adding steel particles and incubating to allow PrP binding;    -   (c) washing the steel particles to remove diluted sample; and    -   (d) detecting abnormal PrP captured on the steel particles using        antibody capable of binding said abnormal PrP.

Suitably step (a) comprises diluting the sample with buffer to comprisefinal concentrations of

-   -   (i) 50 mM to 200 mM buffer agent;    -   (ii) 1% to 4% w/v bovine serum albumin; and    -   (iii) 2% to 4% w/v CHAPS.

Suitably step (a) comprises diluting the sample with buffer to comprisefinal concentrations of

-   -   (i) 100 mM buffer agent;    -   (ii) 2% w/v bovine serum albumin; and    -   (iii) 2% w/v CHAPS.

In one embodiment, suitably the sample is blood and is diluted withbuffer in the range 1:1 to 1:100. More suitably the blood sample isdiluted with buffer in the range 1:10 to 1:100. Most suitably the bloodis from a tga20 mouse and the blood sample is diluted with buffer at1:10; or the blood is from a CD1 mouse and the blood sample is dilutedwith buffer at 1:10; or the blood is from a hamster and the blood sampleis diluted with buffer at 1:10; or the blood is from a sheep and theblood sample is diluted with buffer at 1:100; or the blood is from a cowand the blood sample is diluted with buffer at 1:100; or the blood isfrom a human and the blood sample is diluted with buffer at 1:100.

In one embodiment, suitably the sample is urine and is diluted withbuffer in the range 10:1 to 1:5. More suitably the urine sample isdiluted with buffer at 1:1.

Suitably the buffer further comprises protease inhibitors.

Suitably the antibody of step (d) is selected from the group consistingof ICSM10, ICSM18, ICSM33 and ICSM35. More suitably, the sample is froma tga20 mouse and the antibody is ICSM10; or the sample is from a CD1mouse and the antibody is ICSM33; or the sample is from a hamster andthe antibody is ICSM18; or the sample is from a sheep and the antibodyis selected from the group consisting of ICSM10, ICSM18, ICSM33 andICSM35; or the sample is from a cow and the antibody is ICSM18; or thesample is from a human and the antibody is ICSM18.

Suitably step (c) comprises washing the steel particles to removediluted sample and subjecting the steel particles to a heat treatmentfor 5 minutes. More suitably, the sample is from a tga20 mouse and heattreatment is at 50 to 110 degrees Celsius; or the sample is from a CD1mouse and heat treatment is at 50 to 110 degrees Celsius; or the sampleis from a hamster and heat treatment is at 20 to 115 degrees Celsius; orthe sample is from a sheep and heat treatment is at 115 degrees Celsius;or the sample is from a cow and heat treatment is at 120 degreesCelsius; or the sample is from a human and heat treatment is at 50 to115 degrees Celsius.

Suitably the sample is blood and the steel particles comprise AISI 304stainless steel.

Suitably the sample is urine and the steel particles comprise AISI 316stainless steel.

Suitably the buffer agent is Tris.

Suitably the pH is 8.4.

In another aspect, the invention relates to a dry composition comprisingTris:BSA:CHAPS in the weight ratio 1:1.65:1.65.

In another aspect, the invention relates to a solution comprisingTris:BSA:CHAPS in the molar ratio 1:0.003:0.32.

In another aspect, the invention relates to a composition as describedabove or a solution as described above for use in detection of abnormalPrP.

In another aspect, the invention relates to use of a composition asdescribed above or a solution as described above for detection ofabnormal PrP in a sample.

In another aspect, the invention relates to a kit comprising

-   -   (i) composition as described above or a solution as described        above; and    -   (ii) an anti-prion antibody.

In another aspect, the invention relates to a method of aiding thediagnosis of prion infection in a subject, the method comprising

-   -   (a) providing a sample of blood or urine from said subject    -   (b) assaying said sample blood or urine for abnormal PrP as        described above,        wherein detection of abnormal PrP indicates an increased        likelihood of prion infection in the subject.

In another aspect, the invention relates to a method for detecting prioninfection having sensitivity of at least 71% and specificity of at least99.9%.

Preferred Aspects

In one preferred aspect the invention provides a method for detection ofabnormal PrP in a blood sample, said method comprising:

-   -   (a) diluting the blood sample with buffer to comprise final        concentrations of        -   (i) 10 mM to 500 mM buffer agent;        -   (ii) 1% to 10% w/v bovine serum albumin; and        -   (iii) 1% to 8% w/v CHAPS;    -   (b) adding steel particles and incubating to allow PrP binding;    -   (c) washing the steel particles to remove diluted blood sample;        and    -   (d) detecting abnormal PrP captured on the steel particles using        antibody capable of binding said abnormal PrP.

Suitably step (a) comprises diluting the blood sample with buffer tocomprise final concentrations of

-   -   (i) 50 mM to 200 mM buffer agent;    -   (ii) 1% to 4% w/v bovine serum albumin; and    -   (iii) 2% to 4% w/v CHAPS.

Suitably step (a) comprises diluting the blood sample with buffer tocomprise final concentrations of

-   -   (i) 100 mM buffer agent;    -   (ii) 2% w/v bovine serum albumin; and    -   (iii) 2% w/v CHAPS.

Suitably the blood sample is diluted with buffer in the range 1:1 to1:100.

Suitably the blood sample is diluted with buffer in the range 1:10 to1:100.

Suitably the blood is from a tga20 mouse and the blood sample is dilutedwith buffer at 1:10; or the blood is from a CD1 mouse and the bloodsample is diluted with buffer at 1:10; or the blood is from a hamsterand the blood sample is diluted with buffer at 1:1; or the blood is froma sheep and the blood sample is diluted with buffer at 1:100; or theblood is from a human and the blood sample is diluted with buffer at1:100.

Suitably the buffer further comprises protease inhibitors.

Suitably the antibody of step (d) is selected from the group consistingof ICSM10, ICSM18, ICSM33 and ICSM5.

Suitably the blood is from a tga20 mouse and the antibody is ICSM10; orthe blood is from a CD1 mouse and the antibody is ICSM33; or the bloodis from a hamster and the antibody is ICSM18; or the blood is from asheep and the antibody is selected from the group consisting of ICSM10,ICSM18, ICSM33 and ICSM35; or the blood is from a human and the antibodyis ICSM18.

Suitably step (c) comprises washing the steel particles to removediluted blood sample and subjecting the steel particles to a heattreatment for 5 minutes.

Suitably the blood is from a tga20 mouse and heat treatment is at 50 to110 degrees Celsius; or the blood is from a CD1 mouse and heat treatmentis at 50 to 110 degrees Celsius; or the blood is from a hamster and heattreatment is at 20 to 115 degrees Celsius; or the blood is from a sheepand heat treatment is at 60 to 120 degrees Celsius; or the blood is froma human and heat treatment is at 50 to 115 degrees Celsius.

Suitably the steel particles comprise AISI 304 stainless steel.

Suitably the buffer agent is Tris.

Suitably the pH is 8.4.

In another aspect, the invention relates to a dry composition comprisingTris:BSA:CHAPS in the weight ratio 1:1.65:1.65.

In another aspect, the invention relates to a solution comprisingTris:BSA:CHAPS in the molar ratio 1:0.003:0.32.

In another aspect, the invention relates to a composition as describedabove or a solution as described above for use in detection of abnormalPrP.

In another aspect, the invention relates to use of a composition asdescribed above or a solution as described above for detection ofabnormal PrP in a sample.

In another aspect, the invention relates to a kit comprising

-   -   (i) composition as described above or a solution as described        above; and    -   (ii) an anti-prion antibody.

In another aspect, the invention relates to a method of aiding thediagnosis of prion infection in a subject, the method comprising

-   -   (a) providing a blood sample from said subject    -   (b) assaying said blood sample for abnormal PrP as described        above        wherein detection of abnormal PrP indicates an increased        likelihood of prion infection in the subject.

In another aspect, the invention relates to a method for detecting prioninfection having sensitivity of at least 71% and specificity of at least99.9%.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the invention involve manipulation of the high avidity ofabnormal PrP for metal and plastic surfaces using a solid-state matrixfor capture coupled to direct immunodetection of surface bound material.Quantitative assay sensitivity was first determined using a dilutionseries of exogenous spikes of vCJD-infected brain into whole bloodbefore analysing a blind panel of authentic patient blood samples. Thepanel comprised 190 samples, including those from vCJD and sporadic CJDpatients, non-prion neurological disease controls and normal controlblood samples.

An assay using a sample of blood or urine, most suitably blood, withhigh sensitivity and a specificity sufficient for clinical use isprovided. The introduction of such a test would provide a breakthroughin the management of public health risks associated with vCJD and allowthe identification of asymptomatic individuals, mitigating the risk ofiatrogenic transmission and facilitating early referral for specialistcare and entry into future clinical trials.

DEFINITIONS

The term ‘comprises’ (comprise, comprising) should be understood to haveits normal meaning in the art, i.e. that the stated feature or group offeatures is included, but that the term does not exclude any otherstated feature or group of features from also being present.

Prion Protein/PrP/PrP^(Sc)/PrP^(C)

The invention is concerned with the detection of prion infection orprion infectivity.

More precisely, the invention in concerned with the detection ofabnormal PrP or disease associated PrP. Detection of the presence ofabnormal PrP or disease associated PrP is indicative of prion infection.Thus the invention is primarily concerned with the detection of abnormalPrP and the presence of abnormal PrP aids the diagnosis of prioninfection in the subject being analysed. Presence of abnormal PrP may betaken as an indication of prion infection. Abnormal PrP includesPrP^(Sc) within the complete ensemble of abnormal PrP isoforms; thusabnormal PrP comprises PrP^(Sc).

Most suitably the abnormal PrP may be PrP^(Sc).

The abnormal PrP detected may be quantified. Quantities of abnormal PrPdetected may be expressed as masses, or as equivalents of LD₅₀ Units ofinfectivity.

An exemplary or reference sequence for human PrP is given in SEQ ID NO:7.

Sample

The sample may be blood or urine. For convenience, the assay is mostlydescribed in relation to blood. However, unless otherwise indicated, thedisclosures herein may be applied equally to the assay whether thesample is blood or is urine. Regarding two specific factors (dilutionand steel type) there may be advantages to using a specific conditionwhen the sample is blood and a different specific condition when thesample is urine. For example, although all of the steels used herein maybe applied to blood or to urine, there is an advantage to using steelAISI 304 when the sample is blood and there is an advantage to usingsteel AISI 316 when the sample is urine. Thus it can be appreciated thatthe disclosure provided is applied equally to blood or to urine, exceptwhere specifically mentioned otherwise.

It is a key advantage of the invention that detection of abnormal PrP ina sample of blood or urine, most suitably blood, is disclosed. Inparticular this is accomplished whilst avoiding protease treatment. Inparticular this is accomplished whilst avoiding immunoprecipitationsteps. Thus suitably the sample analysed comprises blood or urine, mostsuitably blood.

The sample may be from a subject. The subject is suitably a mammal, mostsuitably a livestock animal or a human, most suitably a human.

The sample of blood or urine, most suitably blood may be collected as apreliminary step of the method of the invention. More suitably themethod of the invention begins with a sample of blood or urine, mostsuitably blood previously collected so that collection of the sample issuitably not a part of the invention. This has the advantage that thesubject need not be present for the methods of the invention.

Suitably the sample of blood or urine, most suitably blood is dilutedfor detection. Dilution of the sample is very important to theinvention. The capture buffer has been carefully developed for dilutingthe sample to provide excellent results. Dilution of the sample overparticular ranges is important to signal generation.

Blood Dilution

Typically blood is diluted 1:1 to 1:100. Most suitably the optimaldilution is chosen according to the species as in the following table:

Blood Dilution (all or nothing response) Species Low High FunctionalMouse (tga20) 1:1 1:100 1:10  Mouse (CD1) 1:1 1:100 1:10  Hamster 1:11:100 1:1  Sheep 1:1 1:100 1:100 Human 1:1   1:10,000 1:100 Cow 1:11:100 1:100

For species not listed in the table, the skilled worker can determinethe optimal dilution by trial and error using the given values as astart point. In example 5 there is provided a protocol for determinationof optimised dilutions.

The volume of blood required may be chosen by the operator according tothe volume(s) available and the processing requirements. A typicalvolume used is 8 μl of whole blood per aliquot/sample.

Without wishing to be bound by theory, the actual volume used is notthought to be critical. For example, doubling the volume used does notnecessarily double the signal detected—the dilution of the blood is moreimportant as explained above.

Blood derivatives such as blood fractions may be used in place of blood.For example, the red cell fraction may be used, the mononucleocytefraction may be used, or the plasma fraction may be used. Typically suchblood fractions perform in the method of the invention, but whole bloodis preferred since it typically provides a stronger signal than bloodfractions.

Urine Dilution

In contrast to blood, higher dilutions of urine such as 1:100 do nottypically give robust signal for urine.

Typically urine is diluted 10:1 to 1:5. More suitably urine is diluted2:1 to 1:2. Most suitably the optimal dilution of urine is 1:1.

If necessary, the skilled worker can determine the optimal dilution bytrial and error using the given values as a start point. In example 5there is provided a protocol for determination of optimised dilutions.

The volume of urine required may be chosen by the operator according tothe volume(s) available and the processing requirements. A typicalvolume used is 400 μl of whole urine per aliquot/sample.

Without wishing to be bound by theory, the actual volume used is notthought to be critical. For example, doubling the volume used does notnecessarily double the signal detected—the dilution of the sample ismore important as explained above.

When the sample comprises urine, most suitably the urine is human urine.

Diagnosis

The invention provides methods for aiding the diagnosis of prioninfection. Such methods are suitably performed on an isolated samplefrom the subject being investigated. Thus, suitably the methods aremethods which may be conducted in a laboratory setting without the needfor the subject to be present. Suitably the methods are carried out invitro i.e. suitably the methods are in vitro methods. Suitably thesamples are in vitro i.e. suitably the samples are in vitro samples.

Capture

According to the invention, abnormal PrP is captured on steel particles.The inventors have found that specific form(s) and/or amount(s) of steelparticles can provide advantageously good results.

Suitably the steel particles used comprise steel powder.

Suitably the steel used is an austenitic stainless steel.

Suitably the steel used is a ‘3-series’ austenitic steel.

Suitably the steel used comprises AISI 316 or AISI 304; most suitablythe steel used comprises AISI 304.

Suitably when the sample is blood the steel particles comprise AISI 304stainless steel.

Suitably when the sample is urine the steel particles comprise AISI 316stainless steel.

AISI is an international standard for the composition of steel. AISI 304and AISI 316 are austenitic stainless steels with the chemicalcompositions:

-   -   AISI 304 chemical composition: C=0.08% max, Mn=2% max, Cr=19%,        Ni=9.5%,    -   AISI 316 chemical composition: C=0.08% max, Mn=2% max, Cr=17%,        Ni=12%, Mo=2.5%

Steels of these compositions from any manufacturers/suppliers may beused in the invention. Most suitably steel is obtained from thecommercial supplier Goodfellow.

Steel can be used within a concentration range of 5 mg to 90 mg ml⁻¹. Itis most effective in the range 10 to 50 mg ml⁻¹ and is optimal at 23 mgml⁻¹.

The assay performance can be improved by preparing the steel before use.Suitably the steel is prepared by washing in aqueous detergent solutionfollowed by washing in water, and then washing in an aqueous alcoholsolution followed by washing in water. Finally the steel particles maybe resuspended in phosphate buffered saline (PBS) ready for use.

An exemplary preparation method is as follows:

Prepare stock solution of 100 mg/ml of new batch of steel powder (AISI304). Wash approximately 3 g in 30 ml 2% triton-x-100 (sigma)/sterileddH2O in a falcon tube for 2 hours at room temperature. Remove liquidand wash powder 5×10 mins in 30 ml sterile ddH2O on rocking platform.Wash steel in 30 ml 70% ethanol and incubate for 10 mins on rockingplatform, then wash again in 3×30 ml sterile ddH2O. Remove smallaliquots of 1 ml into eppendorf tubes. Remove all liquid and weighpowder. Resuspend in PBS to give a final concentration of 100 mg/mlpowder, aliquot into replica eppendorf tubes giving final mass of steelpowder of 23 mg (230 μl of 100 mg/ml stock), then remove all liquid.

Suitably prepared steel particles are not allowed to dry out before use.

Suitably steel particles are used within about 4 days of preparation.

Capture Buffer

The capture buffer comprises three core elements—detergent, buffer agentand background protein. The capture buffer may also contain one or moreoptional elements as discussed below.

Without wishing to be bound by theory, the buffer described herein havebeen arrived at to provide an advantageous balance between the need todisrupt membranes and release PrP, but without being so stringent as todisrupt PrP conformation and/or binding to steel.

Suitably chaotropic agents such as guanidine are not used. Withoutwishing to be bound by theory, it is believed that guanidinium isineffective, which may be due to the chaotropic action releasing boundmaterial from the steel surface. Suitably chaotropic agents such asguanidine are omitted from the methods and compositions of theinvention.

Concentrations referred to are FINAL concentrations in use. This meansfinal concentrations when diluted with the sample of blood or urine,most suitably blood to form the sample being analysed. Most typically astock solution of capture buffer is prepared having correspondinglyhigher concentrations of the constituents so that when it is combinedwith sample of blood or urine, most suitably blood the concentrationsare the final concentrations. For the avoidance of doubt, theconcentrations being discussed are FINAL concentrations (i.e.concentrations when diluted with sample of blood or urine, most suitablyblood) unless otherwise specified.

Detergent

The detergent is suitably a mild detergent such as a detergent suitablefor use in NMR.

Suitably the detergent is a weak zwitterionic detergent. Suitably thedetergent may be selected from any similar class of detergent such assulphobetaine in any of its forms (3-06, 3-08, 3-10, 3-12, 3-14, 3-16),alkyldimethylamine oxides, alkyl glucosides, alkyl maltosides, alkylsulphates, alkyl thioglucosides, Big CHAPS, CHAPSO, Bile acids,digitonin, glucamides, lecithins, lysolecithins, polyoxyethylenes orquaternary ammonium compounds such as CTAB.

The detergent is suitably CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate).

CHAPS is suitably used at between 1% and 8% w/v, being effective atthese concentrations.

More suitably CHAPS is used at between 2% and 4% which has the advantageof working most effectively.

Most suitably CHAPS is used at 2%, which has the advantage of beingoptimal.

Buffering

The capture buffer may comprise buffer agent such astris(hydroxymethyl)aminomethane (Tris) e.g. Tris.Cl or alternativebuffer. In principle it is expected that any buffer would be functional.Phosphate buffered saline (PBS) may be used but Tris buffer offersimproved performance relative to PBS. Suitably the buffer agentcomprises Tris, e.g., Tris.Cl.

Suitably the buffer agent is used at a concentration of 10 to 500 mM.

More suitably the buffer agent is used at a concentration of between 50and 200 mM, which has the advantage of producing best results.

When calculating the concentration of, or how to prepare, the bufferagent such as Tris, account must be taken of the molecular weight and/orthe salt used. For example Tris has a molecular weight of 121.14, but isoften supplied as Tris.Cl/Tris.HCl, and so molecular weights must betaken into account in preparation of the final concentration desired.For example 1g of Tris may need to be adjusted to 1.3 g if usingTris.HCl (since Tris.HCl has a molecular weight of 157.56). Suchadjustments are well within the abilities of the person skilled in theart. Unless otherwise indicated, weights given are for Tris.

The buffer is suitably pH 8.4.

Protein

Background protein is included in the capture buffer.

Suitably the background protein is Bovine Serum Albumin (BSA). Suitablythis is used between 1% and 10% w/v.

More suitably this is used between 1 and 4%, which has the advantage ofoperating best.

Most suitably this is used at 2%, which has the advantage of beingoptimal.

Protease Inhibitors

Optionally protease inhibitors such as commercially available ‘CompleteInhibitors’ are included in the capture buffer. Most suitably theprotease inhibitors are ‘Roche Complete protease inhibitor cocktailstablets’ (Roche Diagnostics GmbH) and are used according to themanufacturer's instructions. This has the advantage of eliminatingprotease treatments which have formed part of prior art methods. Thisalso has the technical advantage of preserving more abnormal PrP signal.It is believed that some abnormal PrP types are in fact susceptible toprotease degradation. Therefore the invention advantageouslyspecifically excludes protease treatment. Suitably the invention relatesto methods from which protease step(s) are omitted. Suitably the methodsof the invention comprise inhibition of protease activity.

Nuclease

Optionally nuclease such as commercially available ‘Benzonase’ isincluded in the capture buffer.

Chelator

Optionally a chelator such as EDTA may be included in the capturebuffer.

DMSO

Abnormal PrP may be aggregated into fibrillar structures. A solvent foramyloid is dimethyl sulphoxide (DMSO) and this may optionally be used asa pre-treatment for samples such as blood samples.

Thus, optionally a DMSO pretreatment may be applied to the sample beforedilution into capture buffer. When optional DMSO pretreatment is used,it does not affect the final dilution ratio into capture buffer. Whenused, optional DMSO pretreatment can be considered as the first step ina two stage dilution. For example, if a final dilution of 1:100 isdesired and an optional DMSO pretreatment is used, then a first step of1:10 with DMSO may be used followed by a second step 1:10 into a finalconcentration of capture buffer may be used.

When used, it is desired to dilute the sample with DMSO first, and thensecondly dilute into capture buffer. Simply adding DMSO to capturebuffer with the sample in a one-step procedure is not desired.

Thus, when used, the dilution step can be carried out as two sub-steps

(a-1) adding DMSO to sample

(a-2) diluting DMSO-sample with buffer to comprise final concentrationsas described.

For example 8 μl of blood can be diluted to 80 ul in 50% v/v DMSO inPBS. This is optionally incubated at room temperature (21 C) for 4 hourswith agitation. The samples are then diluted to final volume of 800 μlin DDA capture buffer containing 15% v/v DMSO. The assay can then beperformed as described.

Stock Solutions of Capture Buffer

As noted above, higher concentrations of buffer may be prepared for useas stock solutions in order to ease preparation of blood/urinedilutions.

For example a final concentration of 100 mM Tris, 2% BSA, 2% CHAPS maybe desired and a 5× stock may be prepared which would comprise 500 mMTris, 10% BSA, 10% CHAPS. Thus the invention relates to buffers havingthese components in fixed ratios, independent of their preciseconcentrations. These have the advantage of facilitating dilutions andsample preparation. Thus for example compositions comprising this molarratio, whether they be a 1×, 2× or any other concentration are providedby the invention.

Solutions

Amount Optimal molar Component Functional Advantageous Optimal ratioBuffer agent 10 to 500 mM 50 to 200 mM 100 mM 1 (e.g., Tris) BSA 1% (150μM) 1% (150 μM) 2% (300 μM) 0.003 to to 10% (1.5 mM) 4% (600 μM) CHAPS1% (16 mM) 2% (32 mM) 2% (32 mM)  0.32 to to 8% (130 mM) 4% (65 mM)

Dry Compositions

Amount (to make 1 litre solution) Component Functional AdvantageousOptimal Buffer agent 1.21 g to 60.6 g  6.06 g to 24.23 g 12.1 g  (e.g.,Tris MWt 121.14) BSA  10 g to 100 g 10 g to 40 g 20 g CHAPS 10 g to 80 g20 g to 40 g 20 g

Dry Ratios

Ratio Component Functional Advantageous Optimal Buffer agent 1 1 1(e.g., Tris MWt 121.14) BSA 0.17 to 83 0.41 to 6.6 1.65 CHAPS 0.17 to 660.83 to 6.6 1.65

Heat Treatment

A heat treatment is optionally applied to the steel particles afterwashing and before detection. This has advantage of exposing(‘retrieving’) epitopes on the captured PrP and thereby making it moreamenable to detection by antibody.

It is possible that a chemical treatment could be used to exposeepitopes on the captured PrP. However, guanidinium is ineffective, whichmay be due to the chaotropic action releasing bound material from thesteel surface. Therefore it is preferred that the optionalexposing/retrieving epitopes step is performed as a heat treatment.

The duration of the heat treatment is typically 5 minutes.

The heat treatment may be applied by any suitable means known in the artsuch as by water bath (for temperatures up to 100 degrees Celsius), oilbath, heating block, or any other device.

The heat treatment may be optimised depending on the species of PrPbeing detected (i.e. the species from which the sample of blood orurine, most suitably blood is derived). The table below providesexemplary values. Values for species not listed may be determined bytrial and error using these values as a start point.

Temperature (not essential, provides enhancement) Species Low HighOptimal Mouse (tga20) 50 110 110 Mouse (CD1) 50 110 65 Hamster 20 115 20Sheep 60 120 120 Human 50 115 110 Cow 90 120 120

Detection

Detection of the actual abnormal PrP captured on the steel particles canin principle be carried out by any suitable method known in the art.

The term ‘antibody’ should be understood to be an especially suitablereagent or embodiment—it must be noted that antibody fragments, sc-Fv,fused or humanised antibodies or other antibody-derived reagents oraffybodies or aptamer-type binding reagents may be used if desired. Theimportant element is to have a reagent which can selectively,specifically bind abnormal PrP so that the presence or absence orabnormal PrP can be read out from the steel particles.

Thus the precise mode of detection of the abnormal PrP bound to thesteel particles is a matter for the skilled worker. An exemplaryapproach is to contact the steel particles with a primary antibodycapable of binding abnormal PrP, and incubating to allow binding. Excessantibody is then washed away. Binding of this primary antibody may thenbe detected e.g. by use of secondary antibody with chemiluminescence orsimilar.

Suitably the primary antibody is biotinylated which allows anyavidin-based secondary reagent to be used for detection. However, ifdesired the primary antibody may equally be conjugated to an enzyme fordetection such as horseradish peroxidise (HRP) or other well knownmoiety.

In a preferred embodiment detection is by application of one or more ofthe primary antibodies ICSM10, ICSM18, ICSM33 or ICSM35 to the steelparticles. The ICSM antibodies are publicly availably e.g. from D-GenLtd (UK).

Suitably the antibody or antibody derived detection reagent comprisesCDRs of ICSM10, ICSM18, ICSM33, or ICSM35 as appropriate. I.E. suitablythe antibody or antibody derived detection reagent comprises CDR aminoacid sequence as shown herein.

A designation of “B” following an antibody indicates that it isbiotinylated. For example, ICSM10B means ICSM10 biotinylated antibody.

ICSM10 (D-Gen Product No. 0130-01001) is a monoclonal anti-prion protein(PrP); purified mouse immunoglobulin. The antibody isotype is IgG1κ.ICSM10 specifically reacts with human native and denatured PrPC anddenatured PrPSc. The antibody also reacts with sheep, mouse, hamster andbovine PrP. By Western blotting and immunoprecipitation ICSM10 does notbind to diglycosylated PrP; binding only un-glycosylated PrP and asubset of mono-glycosylated PrP. The mono-glycosylated PrP detected byICSM10 has a higher relative molecular mass than detected by ICSM3(Beringue et al., 2003 for mouse PrP).

The detection reagent of the present invention may comprise one or moreantibodies or antibody fragments capable of binding abnormal PrP,mimetics thereof or small molecule(s) capable of binding abnormal PrP orcombinations thereof. Preferably the detection reagent of the inventionis an antibody or fragment thereof, preferably a monoclonal antibody orfragment thereof. Preferably the agent of the invention comprises anantibody or antibody fragment capable of binding abnormal PrP, such asICSM10, ICSM18, ICSM33 or ICSM35 antibody or a fragment thereof.

Suitably the antibody comprises at least the CDRs of one or moreantibodies shown in the sequence listing.

Advantageously when the agent is an antibody, said antibody is ahumanised antibody. Humanisation of antibodies is well known in the artand can be easily accomplished by the skilled worker. For example, anantibody may be humanised with reference to the sequences encoding theCDRs presented herein. In this regard,

-   -   SEQ ID NO: 1 corresponds to ICSM35VH;    -   SEQ ID NO: 2 corresponds to ICSM35VK;    -   SEQ ID NO: 3 corresponds to ICSM18VH;    -   SEQ ID NO: 4 corresponds to ICSM181c.    -   SEQ ID NO: 5 corresponds to ICSM33 VH.    -   SEQ ID NO: 6 corresponds to ICSM33 VK.

Moreover, reference is made to FIG. 4 to 8 which show the CDR and otherkey sequences for exemplary antibodies.

Guidance regarding humanisation may be found for example in theliterature as published by Greg Winter et al., and techniques for themanipulation and production of recombinant antibodies may be found inHarlow and Lane ‘Antibodies—A Laboratory Manual’, Cold Spring Harbourpress.

In one embodiment, the antibodies (or fragments) may advantageously behumanised by manufacture of chimaeric antibodies.

In another embodiment, the antibodies (or fragments) may advantageouslybe CDR-grafted.

In another embodiment, the antibodies (or fragments) may advantageouslybe fully humanised to the extent that the technology permits.

In a preferred embodiment this primary antibody is biotinylated. Morepreferably, this is deliberately over-biotinylated e.g. to approximately14× (i.e. approx 14 biotins per antibody molecule). This may be easilyaccomplished by conventional biotinylation but using approx. 10× theusual amount of biotin precursor/biotin reagent.

The invention may be applied to any species susceptible to prioninfection.

For example the invention may also be applied to bovine species such asdairy or beef cattle; capreoline species such as Capreolus or Cervidae(e.g., elk or venison deer); or any of the species in the tables herein.

Especially advantageous primary antibodies for detection of abnormal PrPfor particular species are shown in the following table. Others may beselected by trial and error with reference to the examples section.

Antibody Species Functional Non-Functional Optimal Mouse (tga20) ICSM10ICSM18, ICSM33, ICSM10 ICSM35 Mouse (CD1) ICSM33 ICSM10, ICSM18, ICSM33ICSM35 Hamster Not determined Not determined ICSM18 Sheep ICSM10,ICSM18, None ICSM33 ICSM33, ICSM35 Human ICSM18 ICSM10, ICSM33, ICSM18ICSM35 Cow ICSM18, ICSM35 ICSM10, ICSM33 ICSM18

In more detail, method for detection of abnormal PrP in a sample ofblood or urine, most suitably blood may be carried out as follows:

-   -   (a) diluting the sample of blood or urine, most suitably blood        with buffer to comprise final concentrations of        -   (i) 10 mM to 500 mM buffer agent;        -   (ii) 1% to 10% w/v bovine serum albumin; and        -   (iii) 1% to 8% w/v CHAPS.    -   (b-i) adding steel particles        -   Optionally the steel particles may be prepared as follows:            Prepare stock solution of 100 mg/ml of new batch of steel            powder (AISI 304). Wash approximately 3 g in 30 ml 2%            triton-x-100 (sigma)/sterile ddH₂O in a falcon tube for 2            hours at room temperature. Remove liquid and wash powder            5×10 mins in 30 ml sterile ddH₂O on rocking platform. Wash            steel in 30 ml 70% ethanol and incubate for 10 mins on            rocking platform, then wash again in 3×30 ml sterile ddH₂O.            Remove small aliquots of 1 ml into eppendorf tubes. Remove            all liquid and weigh powder. Resuspend in PBS to give a            final concentration of 100 mg/ml powder, aliquot into            replica eppendorf tubes giving final mass of steel powder of            23 mg (230 μl of 100 mg/ml stock), then remove all liquid.        -   Ideally the 23 mg ‘dry’ steel particle aliquot is combined            with a final volume of sample (blood-buffer or            urine-buffer mix) of 800 μl.        -   In practice the 23 mg steel has a ‘dry’ volume of approx. 25            μl. Thus in practice the total volume of the sample            (blood-buffer or urine-buffer mix) with the steel added is            approx. 825 μl. It should be noted that ‘dry’ means merely            that the excess liquid has been removed; it does not infer            that the prepared steel is dried; in fact it is better to            avoid the drying of the prepared steel, for example by            drawing off the liquid just before adding the sample or by            covering the steel (e.g. by placing a lid on the container)            after the liquid has been drawn off so that it does not dry            out.    -   (b-ii) incubating to allow PrP binding        -   This may be done by incubating the steel powder with each            sample for at 18° C. o/n at 650 rpm on a thermomixer.    -   (c) washing the steel particles to remove diluted sample;        -   This washing procedure may be done by capturing steel powder            samples with magnetic block and removing supernatant;            washing steel powder samples 2×1 ml PBS/0.05% tween            capturing each time on magnetic block. Vortex samples in            wash buffer and spin briefly. Wash a further 3×1 ml            PBS/0.05% tween capturing each time on magnetic block. Spin            samples briefly and remove all liquid.        -   At this stage, an optional heat treatment may be used to            improve detection. If used, the heat treatment step may be            carried out as follows: Heat treat all samples of steel            powder at appropriate temperature (see table) for 5 minutes            on a heat block. Allow samples to cool for 3 minutes.    -   (d) detecting abnormal PrP captured on the steel particles using        antibody capable of binding said abnormal PrP.        -   The detection step may be accomplished by any suitable            method known in the art.

For example, it may be carried out as follows: incubate steel powdersamples with 50 μl of detection reagent such as primary antibody capableof binding abnormal PrP (e.g. with an appropriate ICSM antibody-seetable) prepared in PBS/1% tween at 1 μg/ml; for 1 hour at 37° C. and 750rpm on a thermomixer; wash steel powder samples 1×1 ml PBS/0.05% tweencapturing each time on magnetic block. Add 1 ml PBS/0.05% tween thenvortex samples in wash buffer and spin briefly. Remove buffer and wash afurther 1×1 ml PBS/0.05% tween capturing each time on magnetic block.Spin briefly and remove all liquid. Incubate steel powder samples with50 μl of Neutravidin-HRP (Pierce) prepared in PBS/1% tween at 1:100,000for 45 minutes at 37° C. and 750 rpm on a thermomixer. Prepare a serialdilution series of secondary detection reagent such as secondaryantibody (1:100,000, 1:1 million and 1:10 million). Wash steel powdersamples 1×1 ml PBS/0.05% tween capturing each time on magnetic block.Add 1 ml PBS/0.05% tween then vortex samples in wash buffer and spinbriefly. Remove buffer and wash a further 1×1 ml PBS/0.05% tweencapturing each time on magnetic block. Mix equal volumes of SupersignalELISA femto chemiluminescent substrate (Thermo Scientific). Add 60 μl ofchemiluminescent substrate to steel powder aliquots, mix thoroughly bypipetting and aliquot into 3×20 μl in 3 replica wells of black ELISAplates (Greiner). To 2 replica wells add 20 μl of each dilution of thesecondary antibody dilution series. Add a further 80 μl per well ofSupersignal ELISA femto chemiluminescent substrate. Place plate intoTecan M1000 plate reader immediately. Shake for 60 seconds on platereader then read plate on luminescence, attenuation automatic settings.

Combinations

The methods of the invention may advantageously be combined e.g. withtonsil biopsy and/or with MRI scan. For example if a subject isindicated as having an increased likelihood of infection by a method ofthe invention, then it may be desired to perform a tonsil biopsy and/orMRI scan in order to confirm the diagnosis.

Application to Urine

The assay steps when urine is the sample are the same as the assay stepswhen blood is the sample. The only difference is the dilution. Detailsare given for preferred blood dilutions and preferred urine dilutionsseparately herein. There are also advantages to different steels forblood and for urine, although each of the steels disclosed herein workswith both sample types.

To the knowledge of the inventors, abnormal PrP has never been detectedin urine before the present invention.

Numerous commercial hormone products are prepared (purified) from urine.Therefore testing of urine is commercially desirable. For example, inCanada approx. 300,000 patients per year receive hormone productspurified from urine. This is a further clear industrial application forthe invention when the sample is urine.

INDUSTRIAL APPLICATION

The invention provides a blood or urine, most suitably blood, basedassay for the detection of prion infection such as vCJD prion infection.

The invention finds application in aiding clinical diagnostic testing incases of neurological disease where prion disease forms part of thedifferential diagnosis.

The methods of the invention may be used in a high throughput format forexample for use by the UK National Blood Service and/or other similarinternational agencies.

The invention finds application in pre-surgical screening of patients toreduce or eliminate the risk of contaminating instruments and medicaldevices with vCJD prions.

The invention may be applied to screening of tissue and/or organdonations.

The invention may be used for detection of various strains/species ofprion disease for elimination from the human food chain, e.g., ChronicWasting Disease, BSE, sheep scrapie.

The methods disclosed may be used in prevalence screening for vCJD inhumans and BSE, CWD and scrapie in animals.

The invention can be used to assist in eradicating animal prion diseasesin commercial herds. The invention may be applied to the testing ofcommercial herds such as of cattle and sheep.

The invention provides a laboratory based test to replace rodentbioassay in many areas of prion research where they are used forsensitivity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows assay sensitivity to exogenous spiked samples: A serialdilution of vCJD-infected brain homogenate was prepared in whole humanblood. A baseline control of a high concentration of normal human brain(106-fold dilution) in whole human blood was used to establish a cut-offthreshold. Dilutions of up to 10-10 of vCJD-infected brain exogenouslyspiked into whole human blood could be detected at a signal level overthree standard deviations above the mean value for a normal brain spike.Data shown is a mean of six replicates and is expressed as a ratiorelative to the cut-off value (dashed line). The two tailed p value forcomparison of 10-10 vCJD and 10-6 normal brain signals is <0.0001(unpaired t test with Welch correction).

FIG. 2 shows discrimination of endogenous vCJD patient blood from normalcontrols: Data shown are the mean chemiluminescent signal (arbitraryunits) of 14 blood samples from unique, confirmed vCJD patients comparedto the mean signal from 100 normal controls. Error bars represent thestandard deviation from the mean signals. The two tailed p value forcomparison of vCJD and normal blood samples is <0.0001 (unpaired t testwith Welch correction).

FIG. 3 shows testing a blind panel of 190 samples: A blind panel of 190blood samples comprising 21 vCJD infected patients, 100 healthy normalcontrols, and 69 other neurological disease controls were tested in twoindependent assays. Data are shown as the chemiluminescent signal ratiorelative to a cut-off determined from the mean+3×SD of normal controls.Samples shown in grey had a ratio less than 1 and were therefore scoredas negative. Panels (A) and (B) represent data from individual assayruns of blind samples. Red bars represent samples with a ratio greaterthan 1 relative to cut off and scored as reactive. Panel (C) is thecombined ratio data of each blind panel sample relative to cut off.Green and blue bars represent samples scored as single reactive ineither assay run 1 or assay run 2, respectively. Red bars representsamples scored as repeat reactive in both assays 1 and 2 and were hencescored as positive.

FIG. 4 to FIG. 8 show annotated antibody sequences. Note: boxed residuesare CDRs; unboxed, bold residues are the start of the constant region;regions that are neither leaders, constant regions, nor CDRs are definedas framework sequence. FIG. 4 presents the amino acid sequences ofICM33VH (SEQ ID NO:5) and ICM33VK (SEQ ID NO:6). FIG. 5 shows the DNAand amino acid sequences of ICSM35VH (SEQ ID NO:1 and NO:8,respectively). FIG. 6 shows the DNA and amino acid sequences of ICSM35VK(SEQ ID NO:2 and NO:9, respectively). FIG. 7 presents the DNA and aminoacid sequences of ICSM18VH (SEQ ID NO:3 and NO:10, respectively). FIG.8A presents the DNA and amino acid sequences and FIG. 8B shows the DNAsequence of ICSM181c (SEQ ID NO:4 and NO: 11, respectively).

FIG. 9 shows data from sheep.

FIG. 10 shows data from cows.

FIG. 11 shows data when urine is the sample.

FIG. 12 shows data when optional DMSO pretreatment is applied.

FIG. 13 shows data when different volumes of assay material areprepared.

FIG. 14 shows an exemplary amino acid sequence of human PrP (SEQ IDNO:7).

The invention is now described by way of example. These examples areintended to be illustrative, and are not intended to limit the appendedclaims.

EXAMPLES Methods Collection and Storage of Human Blood Samples

These studies were approved by the local research ethics committee ofUniversity College London Institute of Neurology and National Hospitalfor Neurology and Neurosurgery. Blood samples were obtained withconsent, collected in EDTA blood tubes and stored frozen at −70° C.Diagnosis of vCJD was made according to established criteria, e.g.,http://www.advisorybodies.doh.gov.uk/acdp/tseguidanceltseguidance_annexb.pdf.Definitesporadic CJD was diagnosed according to WHO criteria and diagnosis ofprobable sporadic CJD according to published WHO criteria with a highspecificity (35). Normal control blood was obtained as EDTA samples fromthe National Blood Service of England and Wales (NBS).

Assaying Blood Spiked with vCJD and Normal Brain Homogenate:

Steel particles (AISI 304; Goodfellow) were prepared by washing in 2%v/v Triton-X-100 in ddH₂O for 2 hours at room temperature. Steel wascaptured with a magnet and washed 5× with an excess of ddH₂O andsterilised by washing in 70% v/v ethanol before washing again with anexcess of ddH₂O. Aliquots of 23 mg were removed to microfuge tubes,captured with a magnet and the liquid aspirated with a pipette. 5 μl of10% w/v vCJD-infected brain homogenate or normal human brain homogenatewas spiked into 9995 μl of whole human blood. From this a serialdilution series of 10⁻¹ to 10⁻¹⁰ of vCJD-infected brain homogenate intowhole human blood was prepared. Each sample was further diluted 2 foldinto capture buffer [200 mM Tris pH 8.4, 4% w/v BSA, 4% w/v CHAPS,2×Complete protease inhibitors (Roche), 80 Units Benzonase (GradeII,Merck)] for assay. To each aliquot of steel powder, 800 μl of spikedblood/buffer mix was added and incubated at 18° C. with agitationovernight.

Steel was captured on a magnetic rack and the supernatant discarded.Steel was washed 5× with 1 ml PBS+0.05% v/v Tween-20 (PBST). After thefinal wash all liquid was removed and the steel heat treated at 115° C.for 5 minutes. To each tube an aliquot of 50 μl of biotinylated primaryantibody ICSM18 (D-Gen, Ltd) prepared at 1 μg/ml in PBS+1% v/v Tween-20was added and incubated at 37° C. with agitation for 1 hour. Sampleswere washed 3× with 1 ml PBST capturing between washes on a magneticrack. Each sample was then incubated with High SensitivityNeutrAvidin-HRP (Pierce) prepared at a 1:100,000 dilution in PBS+1% v/vTween-20 at 37° C. with agitation for 45 minutes.

Finally samples were washed 3× with 1 ml PBST capturing between washeson a magnetic rack. To each sample 60 μl of SuperSignal ELISA Femtochemiluminescent substrate (Pierce) was added and 20 μl of each steelpowder sample was transferred into 3 replica wells of a black flatbottom ELISA plate (Greiner). Immediately prior to reading the plate afurther 80 μl of SuperSignal ELISA Femto chemiluminescent substrate wasadded per well. A dilution series of 1:100,000 to 1:10 million of theHigh sensitivity NeutrAvidin-HRP (Pierce) was prepared to allow forcorrection in absolute chemiluminescent readings across multiple plates.Plates were scanned using a M1000 plate reader (Tecan).

Assaying of Patient Blood Samples:

Steel particles (AISI 304; Goodfellow) were prepared as detailed above.To each aliquot of steel, 800 μl of a 1:100 dilution of whole humanblood into capture buffer (as above) was added and incubated at 18° C.with agitation overnight. Steel was captured on a magnetic rack and thesupernatant discarded before processing as described above. Plates werescanned using a M1000 plate reader (Tecan) in chemiluminescence mode.

The Blind Panel and Scoring of Samples:

A panel of 190 whole blood samples comprising 21 from vCJD patients, 100normal controls (provided by the NBS), 16 patients with probable sCJD,11 confirmed sCJD cases as well as 69 samples from otherneurodegenerative diseases (25 Alzheimer's Disease, 4 Frontal TemporalDementia (FTD), 6 Familial Alzheimer's Disease (FAD) and 7 neurologicalreferrals to the National Prion Clinic confirmed as not Prion Disease)were prepared as blind samples numbered 1 to 190 by parties independentof the assay and analysis. Each of the blind panel samples were testedtwice in independent assays as described above. Samples were processedand analysed in groups of 19 blind panel samples per 96-well plate witha set of 8 quality control samples containing 6 normal control bloodsamples and 2 vCJD-infected patient blood samples.

Samples were scored as reactive if the ratio of the meanchemiluminescence signal from three replicate wells exceeded a cut-offthreshold determined for each plate. The threshold was set at the meanplus 3× Standard Deviations from the mean of the 6 normal blood sampleson each plate. Thus samples with a ratio of greater than 1 wereconsidered reactive. Samples that were reactive in each of the twoindependent assays were scored as positive samples. On completion oftesting all samples in duplicate the results were declared to anindependent party and the samples decoded.

Example 1 Detection of vCJD Brain Homogenate Spiked into Whole Blood

In order to determine the sensitivity of the assay relative to othermethods we analysed serial dilutions of vCJD brain homogenate dilutedinto whole blood to provide a background diluent as close to endogenouspatient samples as possible. A dilution range of 10⁻⁷ to 10⁻¹⁰ of 10%w/v vCJD brain homogenate was assayed and compared to a high backgroundconcentration (10⁻⁶) of normal brain homogenate (10% w/v) also dilutedin whole blood. Although a non-linear response was seen with respect todilution, vCJD-infected brain homogenate could clearly be distinguishedfrom control even at a 10⁻¹⁰ fold dilution (FIG. 1), a sensitivity morethan 4 logs higher than previously achieved for immunoassay of vCJDtissue (31). A chemiluminescent signal of 1.3×10⁵+/−1.1×10⁴ (mean+/−SD)was obtained with 10⁻¹⁰ dilution of vCJD-infected brain versus9.9×10⁴+/−4.5×10³ for a 10⁻⁶ dilution of normal control brain. Data isexpressed as a ratio relative to cut off (see Materials and Methods)this was 1.17+/−0.1 (mean+/−SD). The difference was highly significantwith a p value of <0.0001 (unpaired t-test, two-tailed with Welchcorrection). The highest dilution of vCJD brain was stilldistinguishable from the normal control when a threshold of mean normalsignals+3× standard deviations was applied.

Example 2 Identification of vCJD Infected Patient Bloods

Initial studies performed using exogenous spikes of vCJD-infected brainhomogenate in whole human blood demonstrated our assay was capable ofdiscriminating between infected and non-infected samples with asensitivity theoretically sufficient to detect infection in vCJD bloodbased upon estimates of titre obtained from rodent models (22;23).However, the biochemical nature of infectivity and abnormal PrPassociated with blood is unknown and the results obtained from exogenousspiking experiments cannot be assumed to apply to authentic patientsamples. To ensure this level of discrimination could be achieved withendogenous blood samples we tested a sub-set of confirmed vCJD patientbloods obtained from the National Prion Clinic and compared these tonormal control bloods obtained from the NBS (FIG. 2). The samples wereanalysed as groups which had mean chemiluminescent signals that weresignificantly different (p value <0.0001, unpaired t-test, two-tailedwith Welch correction).

Example 3 Blind Panel Analysis

In order to confirm our results and remove any bias from the analysis apanel of 190 samples taken from 21 confirmed vCJD patients (NationalPrion Clinic), 69 patients with other neurological disease and 100normal healthy controls (NBS) were blinded by parties independent to thetesting and analysis. The samples were tested in batches of 19 sampleswith internal controls to determine a cut-off threshold for each plate,samples which gave chemiluminescence signals above the cut-off weredeemed reactive. To eliminate potential false positive reactionsoriginating not from the sample but from contamination or assay errorseach sample was tested twice within independent assay runs (FIG. 3).Only those samples which were repeat reactive in both assays were scoredas positive.

From the panel of 190 samples tested a total of 19 and 22 were reactivein assays 1 and 2 respectively (FIG. 3). A subset of 15 of those samplesgave signals above the cut-off threshold in both assays and hence werescored positive. Subsequently all 15 positives were decoded as samplesobtained from vCJD patients demonstrating an assay sensitivity of 71%(15/21).

Samples from 6 vCJD patients were not identified during testing of theblind panel. Of those samples 3 had been singularly reactive in eitherassay run 1 or 2 (FIG. 3) potentially indicating a low level of abnormalPrP but not sufficient to be distinguished from controls by theapplication of a threshold. The 3 remaining samples were scored asnegative in both of the test runs and were indistinguishable from normalcontrols.

None of the normal controls or other neurological disease controls werescored as positive suggesting a high level of specificity. Superficiallythis could be considered as 100% as none of the controls were scoredpositive. However, it is important to consider the probability of anegative sample reacting in duplicate assays purely by chance andindependent of abnormal PrP content. This rate is provided by thefrequency of single reactive samples in each independent assay run andprovides a realistic estimation of specificity of 99.97%([2/190]_(Assay 1)×[⁶/190]_(Assay 2)).

Example 4 Direct Detection of Infectivity on Surfaces—Heat TreatmentTemperature Determination

(1) Prepare stock solution of 100 mg/ml of new batch of steel powder(AISI 304). Wash approximately 3 g in 30 ml 2% triton-x-100(sigma)/sterile ddH₂O in a falcon tube for 2 hours at room temperature.Remove liquid and wash powder 5×10 mins in 30 ml sterile ddH₂O onrocking platform. Wash steel in 30 ml 70% ethanol and incubate for 10mins on rocking platform, then wash again in 3×30 ml sterile ddH₂O.

(2) Remove small aliquots of 1 ml into eppendorf tubes. Remove allliquid and weigh powder. Resuspend in PBS to give a final concentrationof 100 mg/ml powder, aliquot into replica eppendorf tubes giving finalmass of steel powder of 23 mg (230 μl of 100 mg/ml stock), then removeall liquid.

(3) Prepare buffer for dilution of bloods so that final concentration ofbuffer will be [100 mM Tris pH8.4+2% BSA+2% CHAPS+1×completeinhibitors+40 units benzonase (grade II)]

(4) Prepare bloods in the above buffer at required dilution so thatfinal volume of blood buffer mix is 800 μl.

(5) Add 800 μl of each sample to steel powder aliquots.

(6) Incubate steel powder with bloods for at 18° C. o/n at 650 rpm on athermomixer.

(7) Capture steel powder samples with magnetic block and removesupernatant.

(8) Wash steel powder samples 2×1 ml PBS/0.05% tween capturing each timeon magnetic block. Vortex samples in wash buffer and spin briefly. Washa further 3×1 ml PBS/0.05% tween capturing each time on magnetic block.Spin samples briefly and remove all liquid.

(9) Heat treat all samples of steel powder at 4° C., 20° C., 60° C., 70°C., 80° C., 90° C., 100° C., 110° C. or 120° C. for 5 minutes on a heatblock.

(10) Allow samples to cool for 3 minutes.

(11) Incubate steel powder samples with 50 μl of determined ICSMantibody prepared in PBS/1% tween at 1 μg/ml; for 1 hour at 37° C. and750 rpm on a thermomixer.

(12) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(13) Spin briefly and remove all liquid.

(14) Incubate steel powder samples with 50 μl of Neutravidin-HRP(Pierce) prepared in PBS/1% tween at 1:100,000 for 45 minutes at 37° C.and 750 rpm on a thermomixer.

(15) Prepare a serial dilution series of secondary antibody (1:100,000,1:1 million and 1:10 million).

(16) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(17) Mix equal volumes of Supersignal ELISA femto chemiluminescentsubstrate (Thermo Scientific)

(18) Add 60 μl of chemiluminescent substrate to steel powder aliquots,mix thoroughly by pipetting and aliquot into 3×20 μl in 3 replica wellsof black ELISA plates (Greiner).

(19) To 2 replica wells add 20 μl of each dilution of the secondaryantibody dilution series.

(20) Add a further 80 μl per well of Supersignal ELISA femtochemiluminescent substrate. Place plate into Tecan M1000 plate readerimmediately. Shake for 60 seconds on plate reader then read plate onluminescence, attenuation automatic settings.

Example 5 Direct Detection of Infectivity on Surfaces—DilutionDetermination

(1) Prepare stock solution of 100 mg/ml of new batch of steel powder(AISI 304). Wash approximately 3 g in 30 ml 2% triton-x-100(sigma)/sterile ddH₂O in a falcon tube for 2 hours at room temperature.Remove liquid and wash powder 5×10 mins in 30 ml sterile ddH₂O onrocking platform. Wash steel in 30 ml 70% ethanol and incubate for 10mins on rocking platform, then wash again in 3×30 ml sterile ddH₂O.

(2) Remove small aliquots of 1 ml into eppendorf tubes. Remove allliquid and weigh powder. Resuspend in PBS to give a final concentrationof 100 mg/ml powder, aliquot into replica eppendorf tubes giving finalmass of steel powder of 23 mg (230 μl of 100 mg/ml stock), then removeall liquid.

(3) Prepare buffer for dilution of bloods as follows so that finalconcentration of buffer will be [100 mM Tris pH8.4+2% BSA+2%CHAPS+1×complete inhibitors+40 units benzonase (grade II)]:

-   -   (a) Prepare 100 ml of [200 mM Tris pH8.4+4% BSA+4% CHAPS+4        complete inhibitor tablets+160 units benzonase]    -   (b) Prepare 50 ml of [110 mM Tris pH8.4+2.2% BSA+2.2%        CHAPS+complete inhibitors+44 units benzonase] by taking 27.5 ml        of your 200 mM Tris buffer prepared in (a) and adding 22.5 ml        water    -   (c) Prepare 50 ml of [101 mM Tris pH8.4+2.04% BSA+2.04%        CHAPS+complete inhibitors+40 units benzonase] by taking 25.3 ml        of your 200 mM Tris buffer prepared in (a) and adding 24.7 ml        water.

(4) Prepare bloods as follows:

-   -   (i) Add 400 μl of blood to 400 μl of 200 mM Tris containing        buffer (a) this is 1:1 blood buffer mix    -   (ii) Add 80 μl of blood to 720μl of 110 mM Tris containing        buffer (b) this is 1:10 blood buffer mix    -   (iii) Add 8 μl of blood to 792 μl of 101 mM Tris containing        buffer (c) this is 1:100 blood buffer mix

(5) Add 800 μl of each sample to steel powder aliquots.

(6) Incubate steel powder with bloods for at 18° C. o/n at 650 rpm on athermomixer.

(7) Capture steel powder samples with magnetic block and removesupernatant.

(8) Wash steel powder samples 2×1 ml PBS/0.05% tween capturing each timeon magnetic block. Vortex samples in wash buffer and spin briefly. Washa further 3×1 ml PBS/0.05% tween capturing each time on magnetic block.Spin samples briefly and remove all liquid.

(9) Heat treat all samples of steel powder at determined temperature for5 minutes on a heat block.

(10) Allow samples to cool for 3 minutes.

(11) Incubate steel powder samples with 50 μl of determined ICSMantibody prepared in PBS/1% tween at 1 μl g/ml; for 1 hour at 37° C. and750 rpm on a thermomixer.

(12) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(13) Spin briefly and remove all liquid.

(14) Incubate steel powder samples with 50 μl of Neutravidin-HRP(Pierce) prepared in PBS/1% tween at 1:100,000 for 45 minutes at 37° C.and 750 rpm on a thermomixer.

(15) Prepare a serial dilution series of secondary antibody (1:100,000,1:1 million and 1:10 million).

(16) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(17) Mix equal volumes of Supersignal ELISA femto chemiluminescentsubstrate (Thermo Scientific)

(18) Add 601 of chemiluminescent substrate to steel powder aliquots, mixthoroughly by pipetting and aliquot into 3×20 μl in 3 replica wells ofblack ELISA plates (Greiner).

(19) To 2 replica wells add 20 μl of each dilution of the secondaryantibody dilution series.

(20) Add a further 801 per well of Supersignal ELISA femtochemiluminescent substrate. Place plate into Tecan M1000 plate readerimmediately. Shake for 60 seconds on plate reader then read plate onluminescence, attenuation automatic settings.

Example 6 Direct Detection of Infectivity on Surfaces—AntibodyDetermination

(1) Prepare stock solution of 100 mg/ml of new batch of steel powder(AISI 304). Wash approximately 3 g in 30 ml 2% triton-x-100(sigma)/sterile ddH2O in a falcon tube for 2 hours at room temperature.Remove liquid and wash powder 5×10 mins in 30 ml sterile ddH2O onrocking platform. Wash steel in 30 ml 70% ethanol and incubate for 10mins on rocking platform, then wash again in 3×30 ml sterile ddH2O.

(2) Remove small aliquots of 1 ml into eppendorf tubes. Remove allliquid and weigh powder. Resuspend in PBS to give a final concentrationof 100 mg/ml powder, aliquot into replica eppendorf tubes giving finalmass of steel powder of 23 mg (23001 of 100 mg/ml stock), then removeall liquid.

(3) Prepare 50 ml buffer for dilution of bloods as follows so that finalconcentration of buffer will be [100 mM Tris pH8.4+2% BSA+2%CHAPS+1×complete inhibitors+40 units benzonase (grade II)]:

(4) Add 8001 of each sample to steel powder aliquots.

(5) Incubate steel powder with bloods for at 18° C. o/n at 650 rpm on athermomixer.

(6) Capture steel powder samples with magnetic block and removesupernatant.

(7) Wash steel powder samples 2×1 ml PBS/0.05% tween capturing each timeon magnetic block. Vortex samples in wash buffer and spin briefly. Washa further 3×1 ml PBS/0.05% tween capturing each time on magnetic block.Spin samples briefly and remove all liquid.

(8) Heat treat all samples of steel powder at determined temperature for5 minutes on a heat block.

(9) Allow samples to cool for 3 minutes.

(10) Incubate steel powder samples with 50 μl of either ICSM10B,ICSM18B, ICSM33B, or ICSM35B antibody prepared in PBS/1% tween at 1μg/ml; for 1 hour at 37° C. and 750 rpm on a thermomixer.

(11) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(12) Spin briefly and remove all liquid.

(13) Incubate steel powder samples with 50 μl of Neutravidin-HRP(Pierce) prepared in PBS/1% tween at 1:100,000 for 45 minutes at 37° C.and 750 rpm on a thermomixer.

(14) Prepare a serial dilution series of secondary antibody (1:100,000,1:1 million and 1:10 million).

(15) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(16) Mix equal volumes of Supersignal ELISA femto chemiluminescentsubstrate (Thermo Scientific)

(17) Add 60 μl of chemiluminescent substrate to steel powder aliquots,mix thoroughly by pipetting and aliquot into 3×20 μl in 3 replica wellsof black ELISA plates (Greiner).

(18) To 2 replica wells add 20 μl of each dilution of the secondaryantibody dilution series.

(19) Add a further 80 μl per well of Supersignal ELISA femtochemiluminescent substrate. Place plate into Tecan M1000 plate readerimmediately. Shake for 60 seconds on plate reader then read plate onluminescence, attenuation automatic settings.

Example 7 Direct Detection of Infectivity on Surfaces—General Protocol

(1) Prepare stock solution of 100 mg/ml of new batch of steel powder(AISI 304). Wash approximately 3 g in 30 ml 2% triton-x-100(sigma)/sterile ddH2O in a falcon tube for 2 hours at room temperature.Remove liquid and wash powder 5×10 mins in 30 ml sterile ddH2O onrocking platform. Wash steel in 30 ml 70% ethanol and incubate for 10mins on rocking platform, then wash again in 3×30 ml sterile ddH2O.

(2) Remove small aliquots of 1 ml into eppendorf tubes. Remove allliquid and weigh powder. Resuspend in PBS to give a final concentrationof 100 mg/ml powder, aliquot into replica eppendorf tubes giving finalmass of steel powder of 23 mg (230 μl of 100 mg/ml stock), then removeall liquid.

(3) Prepare buffer for dilution of bloods so that final concentration ofbuffer will be [100 mM Tris pH8.4+2% BSA+2% CHAPS+1×completeinhibitors+40 units benzonase (grade II)]

(4) Prepare bloods (blood samples) in the above buffer at requireddilution so that final volume of sample (blood buffer mix) is 800 μl.

(5) Add 800 μl of each sample to steel powder aliquots.

(6) Incubate steel powder with each blood sample for at 18° C. o/n at650 rpm on a thermomixer.

(7) Capture steel powder samples with magnetic block and removesupernatant.

(8) Wash steel powder samples 2×1 ml PBS/0.05% tween capturing each timeon magnetic block. Vortex samples in wash buffer and spin briefly. Washa further 3×1 ml PBS/0.05% tween capturing each time on magnetic block.Spin samples briefly and remove all liquid.

(9) Heat treat all samples of steel powder at appropriate temperature(see tables above or as determined in example (1) for 5 minutes on aheat block.

(10) Allow samples to cool for 3 minutes.

(11) Incubate steel powder samples with 50 μl of detection reagent suchas primary antibody capable of binding abnormal PrP (e.g. with anappropriate ICSM antibody) prepared in PBS/1% tween at 1 μg/ml; for 1hour at 37° C. and 750 rpm on a thermomixer.

(12) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(13) Spin briefly and remove all liquid.

(14) Incubate steel powder samples with 50 μl of Neutravidin-HRP(Pierce) prepared in PBS/1% tween at 1:100,000 for 45 minutes at 37° C.and 750 rpm on a thermomixer.

(15) Prepare a serial dilution series of secondary detection reagentsuch as secondary antibody (1:100,000, 1:1 million and 1:10 million).

(16) Wash steel powder samples 1×1 ml PBS/0.05% tween capturing eachtime on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples inwash buffer and spin briefly. Remove buffer and wash a further 1×1 mlPBS/0.05% tween capturing each time on magnetic block.

(17) Mix equal volumes of Supersignal ELISA femto chemiluminescentsubstrate (Thermo Scientific)

(18) Add 601. of chemiluminescent substrate to steel powder aliquots,mix thoroughly by pipetting and aliquot into 3×20 μl in 3 replica wellsof black ELISA plates (Greiner).

(19) To 2 replica wells add 20 μl of each dilution of the secondaryantibody dilution series.

(20) Add a further 80 μl per well of Supersignal ELISA femtochemiluminescent substrate. Place plate into Tecan M1000 plate readerimmediately. Shake for 60 seconds on plate reader then read plate onluminescence, attenuation automatic settings.

Discussion of Examples 1 to 7

Our research efforts have focused upon sensitive methods for thedetection of vCJD infection in whole blood as this is not only a sampleeasily obtained for clinical purposes but also the target for protectingthe donated blood supply. Through the use of matrix capture andoptimised immunodetection of captured material we have been able toachieve detection of vCJD brain homogenate diluted 10¹⁰-fold inexogenous spiking experiments (FIG. 1). This is approximately 100,000fold more sensitive than tonsil biopsy and high sensitivity westernblotting (21) currently in use at the National Prion Clinic. It is also10,000 fold beyond the most sensitive immunoassays for vCJD of unknownspecificity (31) and more sensitive than conventional rodent bio-assaysthat typically do not report dilutions of prion-infected brain tissuegreater than 10⁸-fold (39). Whilst this may at first appearcontradictory rodent bioassays report infectious titre and do not detectPrP molecules directly. The number of PrP monomers that constitute aprion or infectious unit is not fully defined and indeed is likely to beheterogeneous (40). However, it is clear a single infectious unitcontains many PrP monomers as an aggregate all of which are potentiallyavailable to our assay allowing sensitivity beyond that previouslyachievable.

As an orphan disease, blood samples from patients with vCJD are scarceand it is impossible to validate assay results with the high numbers ofsamples typically used in such circumstances. However, we have appliedour assay to all the samples available to us, totaling 21, of which 15were positively identified in our blinded panel, yielding a sensitivityof 71%. Whilst this represents a very significant step forward in priondiagnostics it must be interpreted within the context of associatedspecificity. Indeed for consideration as a screening assay of blood andtissue donations specificity must exceed a minimum of 99.9% to avoidlarge numbers of false positive tests resulting in serious ramificationsfor the individual donors and the related health care agencies.

Our panel contained 100 normal blood samples from the NBS in addition to69 neurological disease controls. We included samples obtained frompatients with Alzheimer's Disease as it has been suggested that abnormalPrP deposition may accompany Aβ accumulation in these diseases (41).Other disorders that may form part of the differential diagnosis forprion disease were also included in the panel. Encouragingly, none ofthe neurological disease controls provided any reactions in either ofthe two independent assay runs, single reactive samples being eithervCJD or normal controls. Although no false positive results wererecorded by our assay criteria a predictive specificity for large scalescreening of controls would be approximately 99.97% based on singlereactive sample frequencies observed. This level of specificity would beacceptable in clinical use. This could usefully be confirmed with large(1000+) numbers of negative controls.

The blood samples obtained from vCJD patients have to date been takenfollowing the onset of symptoms as at present it is not possible toidentify individuals who are definitely infected with vCJD but arecurrently asymptomatic. There remains therefore the question of whenduring the pre-clinical silent stage of disease blood is renderedinfectious or differentiable from normal controls. The availability of ablood based assay for determining prion disease infection could make itpossible to identify preclinical patients infected with vCJD and providethe samples necessary for further assay validation. However, this wouldbe further confounded by our lack of knowledge regarding what a positiveassay result would mean in such circumstances and whether a positiveassay result would always result in an individual progressing to aclinical onset.

The invention thus represents a major advance in the ability to detectprion infection and provides an assay forming part of a viable clinicalblood test.

Example 8 Direct Detection of Infectivity on Surfaces—Detailed Protocol

Note: this protocol is sometimes referred to as the DDA protocol.

(1) Prepare stock solution of 100 mg/ml of new batch of steel powder(AISI 304:45 μm particles, Goodfellow Product Code: 028-638-41). Washapproximately 3 g in 30 ml 2% v/v Triton X-100 (Sigma Code: T8787) inHPLC Grade ddH₂O (VWR Code: 83645.320) in a falcon tube for 2 hr at 30°C. Remove liquid and wash powder 5×10 mins in 30 ml HPLC Grade ddH₂O(VWR Code: 83645.320) on rocking platform. Wash steel in 30 ml 70% v/vHPLC grade Ethanol (Fisher Code: E/0665DF/17)/HPLC Grade ddH₂O (VWRCode: 83645.320) and incubate for 1 hr on rocking platform, then washagain in 3×30 ml HPLC Grade ddH₂O (VWR Code: 83645.320) for 10 min each.

(2) Remove small aliquots of 1 ml into eppendorf tubes. Remove allliquid and weigh powder. Resuspend in PBS (Dulbecco's without Calciumand without Magnesium, GIBCO Code: 14190185 or 14190169) to give a finalconcentration of 100 mg/ml powder, aliquot into replica eppendorf tubesgiving final mass of steel powder of 23 mg (230 μl of 100 mg/ml stock),then remove all liquid. NB—DO NOT ALLOW TO COMPLETELY DRY.

(3) Prepare buffer for dilution of bloods as so that final concentrationof buffer will be [100 mM Tris pH8.4+2% BSA+2% CHAPS+1×completeinhibitors+40 units Benzonase]. Dilutions will vary according to speciesand strain. Refer to Table A for exemplary conditions.

i) Tris Sigma Code: T1503 ii) BSA Sigma Code: A7030 iii) CHAPS SigmaCode: C3023 iv) Complete Inhibitors Roche Code: 1169799801 v) BenzonaseMerk Code: 70664-250KUN

(4) Add 800 μl of each sample to steel powder aliquots.

(5) Incubate steel powder with bloods for at 18° C. O/N at 650 rpm on aThermomixer.

(6) Capture steel powder samples with magnetic block and removesupernatant.

(7) Wash steel powder samples 2×1 ml PBS (BDH 10×Stock Box)/0.05% v/vTween-20 (Sigma Code: P7949) capturing each time on magnetic block.Vortex samples in wash buffer and spin briefly. Wash a further 3×1 mlPBS (BDH 10×Stock Box)/0.05% v/v Tween-20 (Sigma Code: P7949) capturingeach time on magnetic block. Spin samples briefly and remove all liquid.NB—DO NOT ALLOW TO COMPLETELY DRY.

(8) Heat shock all samples of steel powder at determined temperature for5 minutes on a heat block. Temperature will vary according to speciesand strain. Refer to Table A for exemplary conditions.

(9) Allow samples to cool for 5 minutes.

(11) Incubate steel powder samples with 50 μl of appropriate ICSMantibody prepared in PBS (Dulbecco's without Calcium and withoutMagnesium, GIBCO Code: 14190185 or 14190169)/1% v/v Tween-20 (SigmaCode: P7949); for 1 hour at 37° C. and 750 rpm on a Thermomixer.Concentration will typically be in the 600 ng/ml to 1 ug/ml range. Thiswill need to determined for each batch of antibody by titration. ICSMantibody will vary according to species and strain. Refer to Table A forexemplary conditions.

(12) Wash steel powder samples 1×1 ml PBS (BDH 10×Stock Box)/0.05% v/vTween-20 (Sigma Code: P7949) capturing each time on magnetic block. Add1 ml PBS (BDH 10×Stock Box)/0.05% v/v Tween-20 (Sigma Code: P7949) thenvortex samples in wash buffer and spin briefly. Remove buffer and wash afurther 1×1 ml PBS (BDH 10×Stock Box)/0.05% v/v Tween-20 (Sigma Code:P7949) capturing each time on magnetic block.

(13) Spin briefly and remove all liquid.

(14) Incubate steel powder samples with 50 μl of Neutravidin-HRP (PierceCode: PN31030) prepared in PBS (Dulbecco's without Calcium and withoutMagnesium, GIBCO Code: 14190185 or 14190169))/1% v/v Tween-20 (SigmaCode: P7949) at 1:100,000 dilution for 45 min at 37° C. and 750 rpm on aThermomixer. NB—Antibody is typically used at 1:100,000 fold dilutionbut this will need to determined by titration for each new batch.

(15) Prepare a serial dilution series of secondary antibody (1:100,000,1:1 million and 1:10 million).

(16) Wash steel powder samples 1×1 ml PBS (BDH 10×Stock Box)/0.05% v/vTween-20 (Sigma Code: P7949) capturing each time on magnetic block. Add1 ml PBS (BDH 10×Stock Box)/0.05% v/v Tween-20 (Sigma Code: P7949) thenvortex samples in wash buffer and spin briefly. Remove buffer and wash afurther 1×1 ml PBS (BDH 10×Stock Box)/0.05% v/v Tween-20 (Sigma Code:P7949) capturing each time on magnetic block.

(17) Mix equal volumes of Supersignal ELISA Femto ChemiluminescentSubstrate (Pierce Code: PN37074).

(18) Add 60 μl of Supersignal ELISA Femto Chemiluminescent Substrate(Pierce Code: PN37074) to steel powder aliquots, mix thoroughly bypipetting and aliquot into 3×20 μl in three replica wells of black ELISAplates (Greiner Code: 655077).

(19) To two replica wells add 20 μl of each dilution of the secondaryantibody dilution series.

(20) Add a further 80 μl per well of Supersignal ELISA FemtoChemiluminescent Substrate (Pierce Code: PN37074). Place plate intoTecan M1000 plate reader immediately. Shake for 60 sec on plate readerthen read plate on luminescence, attenuation automatic settings.

TABLE A Exemplary Conditions Species Dilution Heat Shock Antibody Mouse(Tg20) 1:10 110 ICSM10 Mouse (CD1) 1:10  65 ICSM33 Hamster A 1:10 RTICSM18 Hamster B 1:1  115 ICSM18 Sheep  1:100 120 ICSM33 Human  1:100110 ICSM18 Cow  1:100 120 ICSM18

The designations “Hamster A” and “Hamster B” refer to two differentpreferred embodiments for assaying hamster samples. When the dilution is1:10, advantageously only a RT heat shock is required; conversely whenthe dilution is 1:1 then advantageously a 115 Celsius heat shock isused.

TABLE B Exemplary Reagents Description Quantity Supplier Product CodeSteel Powder AISI 100 g Goodfellow 028-638-41 304, 45 um particlesBovine Serum 50 g Sigma A7030-50g Albumin (Fraction V) CHAPS 25 g SigmaC3023-25g Benzonase 1 ml Merck 70664-250KUN Chemicals Protease Complete20 tablets Roche 1169799801 Diagnostics ELISA Femto 250 ml PiercePN37074 Chemiluminescence (Fisher) Substrate Neutravidin - 500 ul PiercePN31030 Conjugated HRP (Fisher) ELISA Plates 96 1 case (40) Greiner655077 well Black Bio-One Ltd Plate Sealing 1 box (100) SigmaZ369695-100EA film Triton X100 100 ml Sigma T8787-100ML Trizma Buffer 1kg Sigma T1503-1KG (Base) Pure Water 2.5 l VWR 83645.320 (HPLC Grade)Bottled PBS 10 × 500 ml GIBCO 14190169 (500 ml) (Invitrogen) Bottled PBS20 × 100 ml GIBCO 14190185 (100 ml) (Invitrogen) Ethanol 2.5 l FisherE/0665DF/17 Scientific Chloros 5 l Solmedia CHL005 2 ml Screw 5000Sarstedt Ltd 72.692 Cap Eppendorfs Eppendorf 1000 Sarstedt Ltd 65.716Screw Caps Pippette Tips  960 ANACHEM RT-10S (10 ul) Pippette Tips  5 ×1000 Thistle TF-1000-L-R-S-CS (200 ul) Scientific Pippette Tips 5 × 960Thistle TF-200-L-R-S-CS (1000 ml) Scientific Pipette Tips 5 × 960Thistle TF-20-L-R-S-CS (20 ul) Scientific Nunc Tubes  450 Greiner 210261(50 ml) Bio-One Ltd Falcom Tubes 1000 Greiner 188271 (15 ml) Bio-One LtdTween-20 100 ml Sigma P7949-100ml ICSM18 Anti- 1 mg D-Gen Ltd 0130-01810PrP Antibody

Example 9

In this example, the invention is demonstrated for sheep.

The standard DDA protocol (e.g. as in Example 8) using biotinylatedICSM33 as a primary antibody, a dilution of blood 1:100 into capturebuffer and a heat treatment at 120 C was applied to a range of sheepblood samples taken from 8 known uninfected controls and 8 cases ofconfirmed scrapie. All 8 positive samples were distinguishable fromnegative controls. Data are shown in FIG. 9.

Example 10

The standard DDA protocol (e.g. as in Example 8) using biotinylatedICSM18 as a primary antibody, a dilution of blood 1:100 into capturebuffer and a heat treatment at 120 C was applied to a range of cattleblood samples taken from 8 known uninfected controls and 8 cases ofconfirmed BSE. All 8 positive samples were distinguishable from negativecontrols. Data are shown in FIG. 10.

Example 11

In this example, we demonstrate discrimination of vCJD urine fromcontrol samples.

A standard DDA protocol (e.g. as in Example 8) was carried out usingbiotinylated ICSM18 as a primary antibody, a dilution of urine 1:1 intocapture buffer and a heat treatment at 110 C was applied to a panel of12 control urine samples and 4 samples collected from vCJD patients. Thestandard DDA protocol was modified by the use AISI 316 steel in place ofthe standard AISI 304. AISI 316 gives better results when the sample isurine. A cut-off value was calculated as the mean chemiluminescencesignal of the 12 control samples plus 3×standard deviations from themean.

Three of the four vCJD patient samples were considered positive, i.e.had a ratio of greater than 1. N.B. Although AISI 304 steel alsofunctions with urine samples, AISI 316 gives superior results. Data areshown in FIG. 11.

Example 12

In this example, optional DMSO pretreatment is demonstrated.

A potential issue for the methods of the invention is that abnormal PrPis likely to be aggregated into fibrillar structures. A solvent foramyloid is dimethyl sulphoxide (DMSO) and this was investigated as apre-treatment for samples, in this example blood samples.

8 ul of blood was diluted to 80 ul in 50% v/v DMSO in PBS and incubatedat room temperature (21 C) for 4 hours with agitation. The samples werethen diluted to final volume of 800 ul in DDA capture buffer containing15% v/v DMSO. The assay was then performed as described above.

Pre-treatment increases the signal levels obtained from vCJD samples butdoes not affect control samples, thereby advantageously increasing thelevels of differentiation. Data are shown in FIG. 12.

Example 13

A typical high throughput and/or automated assay uses a 96-well format.However, such wells are necessarily of a rather small size. This is anadvantage for sample processing and space considerations, but the smallsize restricts the volume available for analysis.

According to the invention, the assay volume can be varied (such asincreased) since it is a flexible part of the method. However, it isimportant to make any volume increases in conjunction with an increasedamount of steel particles/powder (i.e. matrix). In other words theratios of components of the assay are suitably kept within the rangesdescribed above, but by increasing the amount of each of the individualcomponents, a greater volume can be assayed whilst maintaining theratios of the components within the described ranges.

To demonstrate this, the assay was performed as per the protocoldescribed above (e.g., Example 8) with the exception of:

-   -   the volume of blood analysed, 8, 12 or 16 μl and hence the blood        to capture buffer ratio, i.e., 1:100, 1:67 and 1:50.    -   the concentration of steel particles in the capture buffer; 23,        35 or 46 mg/ml.    -   the chemiluminescence was read in a 24 well plate using 1000 ul        of ELISA femto chemiluminescent substrate.

Increasing the volume of blood used in the assay with a fixed steelconcentration of 23 mg/ml gives no improvement in signal.

A marginal improvement can be seen with 12 μl of blood and increasedsteel concentrations.

A significant enhancement is obtained by increasing the input to 16 μlin conjunction with increasing the steel concentration to 46 mg/ml. Inthis embodiment the input is doubled (8 μl to 16 μl) and the steel isdoubled (23 mg to 46 mg) so the proportions remain the same and thecapture buffer concentrations remain the same; the net effect is toprepare more of the steel-captured-analyte (the PrP) by use of thelarger volumes without altering the core of the method of the invention.Thus the method of the invention can easily be scaled up to largervolumes to suit alternate read-out or assay formats as desired by theskilled worker. Data are shown in FIG. 13.

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All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed aspects and embodiments of the present invention will beapparent to those skilled in the art without departing from the scope ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are apparent tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for detection of abnormal PrP in a sample of urine, saidmethod comprising: (a) diluting the urine sample with buffer to comprisefinal concentrations of (i) 10 mM to 500 mM buffer agent; (ii) 1% to 10%w/v bovine serum albumin; and (iii) 1% to 8% w/v3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);wherein said buffer does not include chaotropic agents or proteinases;(b) adding steel particles and incubating to allow PrP binding; (c)washing the steel particles to remove diluted sample; (d) subjecting thewashed steel particles to a heat treatment; and (e) detecting abnormalPrP captured on the steel particles using antibody or portion thereofcapable of binding said abnormal PrP; wherein said method is practicedwithout use of chaotropic agents or proteinases.
 2. The method accordingto claim 1 wherein step (a) comprises diluting the sample with buffer tocomprise final concentrations of (i) 50 mM to 200 mM buffer agent; (ii)1% to 4% w/v bovine serum albumin; and (iii) 2% to 4% w/v CHAPS.
 3. Themethod according to claim 1, wherein step (a) comprises diluting thesample with buffer to comprise final concentrations of (i) 100 mM bufferagent; (ii) 2% w/v bovine serum albumin; and (iii) 2% w/v CHAPS.
 4. Themethod according to claim 1, wherein the urine sample is diluted withbuffer in the range of 2:1 to 1:2.
 5. The method according to claim 4,wherein the urine sample is diluted with buffer at 1:1.
 6. The methodaccording to claim 1, wherein the buffer further comprises proteaseinhibitors.
 7. The method according to claim 1, wherein the antibody ofstep (e) is selected from the group consisting of ICSM10, ICSM18,ICSM33, ICSM35, and portions thereof.
 8. The method according to claim7, wherein the sample is from a tga20 mouse and the antibody is ICSM10or a portion thereof; or the sample is from a CD1 mouse and the antibodyis ICSM33 or a portion thereof; or the sample is from a hamster and theantibody is ICSM18 or a portion thereof; or the sample is from a sheepand the antibody is selected from the group consisting of ICSM10,ICSM18, ICSM33, ICSM35, and portions thereof; or the sample is from acow and the antibody is ICSM18 or a portion thereof; or the sample isfrom a human and the antibody is ICSM18 or a portion thereof.
 9. Themethod according to claim 1, wherein step (d) comprises subjecting thesteel particles to a heat treatment for 5 minutes.
 10. The methodaccording to claim 9, wherein the sample is from a tga20 mouse and heattreatment is at 50 to 110 degrees Celsius; or the sample is from a CD1mouse and heat treatment is at 50 to 110 degrees Celsius; or the sampleis from a hamster and heat treatment is at 20 to 115 degrees Celsius; orthe sample is from a sheep and heat treatment is at 115 degrees Celsius;or the sample is from a cow and heat treatment is at 120 degreesCelsius; or the sample is from a human and heat treatment is at 50 to115 degrees Celsius.
 11. The method according to claim 10, wherein thesteel particles comprise AISI 316 stainless steel.
 12. The methodaccording to claim 1, wherein the buffer agent is Tris.
 13. The methodaccording to claim 1, wherein the pH is 8.4.
 14. The method according toclaim 1, wherein the urine is human urine.
 15. Use of a dry compositioncomprising Tris:BSA:CHAPS in the weight ratio 1:1.65:1.65 or a solutioncomprising Tris:BSA:CHAPS in the molar ratio 1:0.003:0.32 for detectionof abnormal PrP in a urine sample.
 16. A method of aiding the diagnosisof prion infection in a subject, the method comprising (a) providing asample of urine from said subject; and (b) assaying said sample of urinefor abnormal PrP according to claim 1, wherein detection of abnormal PrPindicates an increased likelihood of prion infection in the subject. 17.The method of claim 10, wherein the sample is from a human and the heattreatment is at 110 degrees Celsius.